Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor

Background and Purpose

Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A₁ receptor for this purpose.

Experimental Approach

We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A₁ receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data.

Key Results

The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate.

Conclusion and Implications

The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A₁ receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well.Table of Links

Intake of added oils and fats among middle-aged French adults: relationships with educational level and region of residence

OBJECTIVE: To describe the relative contribution of 10 created food groups to total fat intake in middle-aged subjects. The relationship of added oil and fat intake with region of residence and educational level was also assessed. DESIGN: Cross-sectional study. SUBJECTS/SETTING: Participants of the French Supplémentation en Vitamines et Minéraux Antioxydants study who completed at least six 24-hour dietary records after inclusion into the study (N=6,572). RESULTS: Added oils and fats were the main source of total fat intake. Animal fat and margarine intakes showed a significant inverse association with educational level, whereas oils with monounsaturated fatty acids (MUFA) were positively associated with education level. Animal fat intake was significantly higher in the western and northern parts of France (54.2% and 50.4%) and lower in the Mediterranean Coast (39.0%). A significant inverse gradient was found with oils with polyunsaturated fatty acids or MUFA in the southwest and the Mediterranean Coast compared with the northern part of France. CONCLUSIONS: We showed a north to south gradient for animal fat intake and the opposite for oils with MUFA and polyunsaturated fatty acid in France. This gradient parallels the known disparities for cardiovascular mortality in this country. This should contribute to adapting dietary guidelines for dietary change in a public health perspective.     

Dietary fiber intake and risk factors for cardiovascular disease in French adults

BACKGROUND: Increased consumption of dietary fiber is widely recommended to maintain or improve health, but knowledge of the relation between dietary fiber sources and cardiovascular disease risk factors is limited. OBJECTIVE: We examined the relation between the source or type of dietary fiber intake and cardiovascular disease risk factors in a cohort of adult men and women. DESIGN: In a cross-sectional study, quintiles of fiber intake were determined from dietary records, separately for 2532 men and 3429 women. Age- and multivariate-controlled logistic models investigated the odds ratios of abnormal markers for quintiles 2-5 of fiber intake compared with the lowest quintile. RESULTS: The highest total dietary fiber and nonsoluble dietary fiber intakes were associated with a significantly (P < 0.05) lower risk of overweight and elevated waist-to-hip ratio, blood pressure, plasma apolipoprotein (apo) B, apo B:apo A-I, cholesterol, triacylglycerols, and homocysteine. Soluble dietary fiber was less effective. Fiber from cereals was associated with a lower body mass index, blood pressure, and homocysteine concentration; fiber from vegetables with a lower blood pressure and homocysteine concentration; and fiber from fruit with a lower waist-to-hip ratio and blood pressure. Fiber from dried fruit or nuts and seeds was associated with a lower body mass index, waist-to-hip ratio, and fasting apo B and glucose concentrations. Fiber from pulses had no specific effect. CONCLUSION: Dietary fiber intake is inversely correlated with several cardiovascular disease risk factors in both sexes, which supports its protective role against cardiovascular disease and recommendations for its increased consumption.     

Consumption of foods rich in flavonoids is related to a decreased cardiovascular risk in apparently healthy French women

A high consumption of flavonoids may lower cardiovascular risk through their antioxidant capacity. This study evaluated the relation between consumption of foods rich in flavonoids and estimated cardiovascular risk. A cross-sectional analysis was performed in 1286 women and 1005 men of the SU.VI.MAX Study (an 8-y trial evaluating the effect of antioxidant supplementation on the incidence of major chronic diseases). Dietary intakes were estimated using six 24-h dietary records collected during the year between the clinical measurement of blood pressure, weight and height and the biological measurement of total serum cholesterol and fasting plasma glucose. The relation between flavonoid rich food consumption and cardiovascular risk factors was evaluated with analyses of covariance and the effect on cardiovascular risk with logistic regression analyses. In women, flavonoid-rich food consumption was inversely related to systolic blood pressure (P = 0.005). No relation between risk factors and flavonoid-rich food consumption was seen in men. Women in the highest tertile of flavonoid-rich food consumption were at lower risk for cardiovascular disease [odds ratio (OR): 0.31; 95%CI: 0.14, 0.68], whereas a positive tendency was seen in men (OR: 1.38; 95%CI: 0.96, 2.00). These results indicate that in women, a high consumption of flavonoid-rich foods may prevent cardiovascular disease.     

Well-characterized garlic-derived materials are not hypolipidemic in APOE*3-Leiden transgenic mice

Garlic is reported to have beneficial effects on risk factors associated with cardiovascular disease, including normalization of plasma lipid levels. However, numerous studies do not support this beneficial effect of garlic on plasma lipids. This contradiction may result from the use of different garlic-derived materials, experimental designs, and/or animal models. The present study investigated the hypolipidemic effect of garlic-derived materials in APOE*3-Leiden mice, a model well suited for drug and dietary intervention studies of hyperlipidemia. APOE*3-Leiden mice were fed a garlic-derived sulfur-rich compound, either allicin (0.29 g.L drinking water(-1)) or diallyldisulfide (0.27 g.kg diet(-1)), or powdered garlic, of either the kwai (42 g.kg diet(-1)) or morado (42 g.kg diet(-1)) variety. The amounts of garlic-derived materials supplied allowed free intake of allicin or allicin equivalents (diallyldisulfide, kwai, or morado) at 44 mg.kg body wt(-1).d(-1). Mice were fed a nonpurified diet for 4 wk, followed by a Western diet for 8 wk, both supplemented with the garlic-derived materials. These diets had no consistent effect on plasma lipids and did not affect lipoprotein profiles, which are markers for whole-body cholesterol synthesis and intestinal sterol absorption. The current data indicate that the postulated effects of garlic on cardiovascular disease are not caused via modulation of plasma lipid levels.     

Dental laboratory work authorization forms: a national survey of regulatory agency requirements

This study reports a survey of the dental licensing agencies in the 50 states, Washington, D.C., and Puerto Rico concerning agency legislation and nomenclature of dental laboratory work orders. Differences in both legislation and nomenclature were found. Dental laboratory work authorization was the title most frequently used by the agencies. Nearly all agencies require a work order written by a licensed dentist to accompany all materials transmitted to a dental laboratory. This along with other sources of information may help to increase uniformity in the content of laboratory work authorization forms, thereby improving quality control in dental restorations made in commercial laboratories.     

Combined effects of fasting and alanine on liver function recovery after cold ischemia

The effect of donor nutritional status on hepatic function recovery after cold ischemia is still debated. We demonstrated previously that a 48-h fast diminished the survival rate of liver-transplanted rats and that the deleterious effect of fasting was prevented by infusion of alanine to the recipient at reperfusion. Whether the duration of fasting influenced the protective effect of alanine and whether this effect was metabolic were not known, and the elucidation of these questions is the aim of this study. The effect on hepatic function recovery of fasting periods of 24 h, 48 h and 72 h prior to cold ischemia were studied in a model of isolated, perfused rat liver. After a cold-ischemic time of 24 h in University of Wisconsin (UW) solution at 4 degrees C, livers were reperfused for 3 h. The combined effect of alanine (8 mM) infusion at liver reperfusion was evaluated for each prior fasting period. The addition of pyruvate (8 mM), a metabolic intermediary of alanine, was only tested in the 72-h fasting group. The evaluation criteria were: liver weight after reperfusion, release of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in the perfusate, bile production, vascular resistance and liver histology after reperfusion. The enzyme release at reperfusion was significantly higher when livers were harvested from rats submitted to a 48-h fast (ALT) or a 72-h fast (ALT, AST, LDH), as compared to those from fed rats. Vascular resistance was increased in 72-h fasted livers. An addition of alanine (8 mM) at reperfusion lowered the release of AST, ALT and LDH. This effect was more obvious when the fasting duration was increased. By contrast, the addition of pyruvate at reperfusion did not improve the recovery of livers submitted to a 72-h fasting period before preservation. A long fasting period is deleterious as compared to feeding; however, this effect can be compensated by infusion of alanine at reperfusion. The mechanism involved is not metabolic. In a clinical setting, the infusion of alanine to the recipient at reperfusion may be a convenient way to compensate for donor undernutrition, especially after a long stay in an intensive care unit.